1. Field of the Invention
The present invention relates to a complex comprising the neurotrophic factor nerve growth factor (NGF) and trk-proto-oncogene protein receptor and a complex comprising the NGF related neurotrophic factors, neurotrophin-3 (NT-3) or brain-derived neurotrophic factor (BDNF) bound to trkB-proto-oncogene protein. The present invention also relates to a method of detecting the presence of NGF, NT-3 and BDNF ligands, and trk and trkB-proto-oncogene receptor proteins.
The present invention further relates to methods of diagnosing and treating conditions of nerve growth disease and regeneration such as Alzheimer's disease and neuroblastoma. In particular, the present method involves detection of the ligand-receptor pairs.
The present invention further relates to a method of detecting other neurotrophic factor receptor/ligand complexes on the basis of structural and functional relatedness to the trk receptor and NGF.
2. Background Information
The development of the vertebrate nervous system is characterized by a series of complex events beginning with an apparently homogeneous neuroepithelium in the early embryo and leading to formation of diverse, highly ordered, and interconnected neural cell types in the adult. Considerable descriptive and experimental evidence has been amassed which points to the existence of limiting diffusible factors that are required for the targeting, survival, and proper synaptic arrangement of neurons (R. W. Oppenheim, In: Studies in Developmental Neurobiology. (Cowan, W. M. ed.), Oxford University press, pp. 74-133, 1981; W. D. Snider and E. M. Johnson, Ann. Neurol. 26:489-506 (1989)). Functional neuronal circuits are sculpted from an initially overabundant production of neurons during development. In the mid term embryo, a process of programmed cell death eliminates a major proportion of the neuron population, leaving behind the appropriate number of neurons required for innervation of target tissues (V. Hamburger and R. Levi-Montalcini, J. Exp. Zool. 111:457-502 (1949); Y.-A. Barde, Neuron 2:1525-1535 (1989)).
The discovery of nerve growth factor (NGF) provided the first direct evidence for the existence of neurotrophic, polypeptide factors (R. Levi-Montalcini and V. Hamburger, J. Exp. Zool. 116:321-362 (1951); R. Levi-Montalcini and P. U. Angeletti, Physiol. Rev. 48:534-569 (1968)). This has been followed by the more recent description of additional neurotrophic factors: BDNF, ciliary neurotrophic factor (CTNF), and NT-3 (for review see W. D. Snider and E. M. Johnson, Ann. Neurol. 26:489-506 (1989); G. Barbin et al., J. Neurochem. 43:1468-1478 (1984); P. C. Maisonpierre et al., Science 247:1446-1451 (1990)). The physiological consequences elicited by NGF in vitro and in vivo have been at the center of research in neurobiology for several decades. Consequently, considerable information is now available about the cell types that respond to NGF in the peripheral and central nervous systems.
NGF is known to play a role in the targeting and survival of sympathetic and neural crest-derived sensory neurons as well as in selected populations of cholinergic neurons in the brain (L. A. Greene and E. M. Shooter, Annu. Rev. Neurosci. 3:353-402 (1980); H. Thoenen and Y.-A. Barde, Physiol. Rev. 60:1284-1335 (1980); H. Gnahn et al., Dev. Brain. Res. 9:45-52 (1983)). It appears that the NGF dependent cholinergic neurons in the basal forebrain correspond to the population of cells that undergo attrition in patients with Alzheimer's disease (F. Hefti, Annals of Neurology, 13:109-110 (1983); Hefti and Wemer, (1986); Johnson and Tanuchi, (1987); P. J. Whitehouse et al., Science 215:1237-1239 (1982)). In vivo studies, in which NGF was injected in the periphery of the mouse embryo trunk, result in enhanced survival of sensory ganglia that are normally targeted for cell death (V. Hamburger et al., J. Neurosci. 1:60-71 (1981); I. B. Black et al., In: Growth Factors and Development, Current Topics in Developmental Biology, Vol. 24 (Nilsen-Hamilton, ed.), pp. 161-192 (1990)).
Exposure of embryos to NGF antibodies, that thereby partially neutralize the function of NGF, results in reduced survival of dorsal root ganglion neurons while injection of NGF antibodies into neonate mice has the principal effect of inhibiting the survival of sympathetic neurons (R. Levi-Montalcini and B. Booker, Proc. Natl. Acad. Sci. USA, 46:373-384 or 384-391 (1960); S. Cohen, Proc. Natl. Acad. Sci. USA, 46:302-311 (1960); E. M. Johnson et al., Science 210:916-918 (1980)).
In vitro, some tumor cell lines of neural origin respond to the presence of NGF by undergoing differentiation along neuronal pathways. PC12 cells, derived from a rat pheochromocytoma, are the best characterized of these cell lines and represent a widely accepted model for NGF-mediated response and for neuronal differentiation (L. A. Greene and A. S. Tischler, Proc. Natl. Acad. Sci. USA 73:2424-2428 (1976)).
Although much is understood about the biology of NGF outside the cell, the mechanisms by which NGF elicits neurotrophic effects within the cell have not been fully resolved. Interaction of NGF with a cell receptor is a requisite step in the transmission of neurotrophic signals within the cell (for review see M. V. Chao, In: Handbook of Experimental Pharmacology, Vol. 95/II Peptide Growth Factors and Their Receptors II (Sporn, M. B. and Roberts, A. B. eds.), Springer-Verlag, Heidelberg, pp. 135-165 (1990)).
A major advance in understanding NGF interactions with the cells was the identification and cloning of a 75 kDa receptor (75kNGF-R) that binds NGF, and is present in NGF-responsive cells. The clones of the gene encoding 75kNGF-R have been characterized from several species (M. V. Chao et al., Science 232:418-421 (1986); M. J. Radeke et al., Nature 325:593-597 (1987)). Unfortunately, the structural and biological properties of 75kNGF-R have provided limited clues about the nature of the NGF signal transduction pathway inside the cell. 75kNGF-R displays the binding properties of a low affinity NGF receptor (Kd.apprxeq.10.sup.-9 M) when expressed in exogenous cell lines and analysis of the intracellular domain has not revealed putative domains of catalytic action (M. V. Chao, (1990)).
The biological responsiveness to NGF, however, is widely held to depend upon interactions with a high affinity binding component implying that other receptor or receptor subunits may be involved in NGF responses. The search for potential second messengers that might transmit NGF signals in PC12 cells has led to recent evidence indicating that activation of tyrosine kinases may represent an early response to the presence of NGF (Maher (1988)). These data implicate tyrosine kinases as candidates in the composition of a high affinity receptor.
Recent experiments suggest that the trk tyrosine kinase gene family fulfills this role (D. R. Kaplan et al., Nature 350:158-150 (1991a) and Science 252:554-558 (1991b); Hempstead et al., Nature 350:678-683 (1991); R. Klein et al., Cell 65:189-197 (1991)). The trk gene family is comprised of two well-characterized genes, trk and trkB (D. Martin-Zanca et al., Nature 319:743-748 (1986); R. Klein et al., EMBO J. 8:3701-3709 (1989), Development 4:845-850 (1990a) and Cell 61:647-656 (1990b); D. S. Middlemas et al., Mol. Cell. Biol. 11:143-153 (1991)), as well as additional uncharacterized members. The human trk oncogene was first identified in the NIH 3T3 transformation focus-forming assay, and other oncogenic alleles have been detected in human colon and papillary thyroid carcinomas (Martin-Zanca et al., (1986); Bongarzone et al., Oncogene 4:1457-1462, (1989)). Molecular cloning and sequencing analysis of the human proto-oncogene revealed a 790 amino acid open reading frame that displays all the recognizable features of a protein tyrosine kinase receptor molecule of 140 kd (gp 140.sup.proto-trk) (Martin-Zanca et al., (1986)). Applicants and others have cloned and studied the mouse trk proto-oncogene and found that expression of this gene is restricted primarily to subpopulations of sensory neural crest-derived neurons (Martin-Zanca et al., Genes Dev. 4:683-694 (1990)) and expression of the trk protein tyrosine kinase receptor gene family is limited exclusively to the murine embryonic and mature nervous system. (The Avian Model in Developmental Biology: From Organism to Genes, Editions du CNRS--pp. 291-302 (1990)). The expression profile seen for trk correlates, at least in part, with the location of specific neurons that depend on NGF for their survival.
The trkB gene also encodes a protein tyrosine kinase receptor that displays strong evolutionary conservation with trk. The respective tyrosine kinase domains share &gt;88% amino acid homology. The extracellular ligand-binding domains also share homology (.about.40%), including conservation of all cystine residues, although some regions have diverged considerably (Klein et al., (1989)); Middlemas et al., (1991)).
In situ RNA expression studies have demonstrated that, like trk, trkB expression is primarily confined to tissues of the nervous system (Klein et al., (1989), (1990), (1990); Martin-Zanca et al., (1990)). However, additional features in the organization and transcription of the trkB gene depart considerably from those previously observed for the trk gene. trkB transcripts are widespread throughout the central and peripheral nervous systems and are not confined to neurons as they can also apparently be found in nonneuronal cells (such as glia and Schwann cells) (Klein et al., (1989) and (1990)). In contrast, trk gene expression is confined to a subpopulation of neural crest-derived sensory neurons in the peripheral nervous system (Martin-Zanca et al., (1990)) and in neurons of the basal forebrain and trigeminal mesencephalic nucleus. Furthermore, the trk gene apparently encodes only a single transcript (3.2 kb), while multiple transcripts ranging from 0.7 to 9.0 kb have been observed when mouse or rat brain mRNAs are hybridized with trkB probes (Klein et al., (1989) and (1990); Middlemas et al., (1991)). The quantity and location of the various-sized trkB transcripts are differentially regulated during embryonic development (Klein et al., (1990)).
Yet another departure between trk and trkB is that the trkB gene encodes at least two distinct molecules (Klein et al., (1990)). One trkB protein product is colinear with the trk receptor and constitutes a tyrosine kinase receptor glycoprotein of 145 kd (gp 145.sup.trkB). The second molecule is identical to gp 145.sup.trkB in the extracellular and transmembrane domains (465 amino acids) but diverges in the intracellular region, lacking a catalytic tyrosine kinase domain and encoding a short cytoplasmic stretch of 23 amino acids; the predicted size of this form is 95 kd (the final 11 carboxy-terminal residues are unique to gp95.sup.trkB (Klein et al., (1990); Middlemas et al., (1991)). Anti-peptide antibodies generated against both forms of the trkB product have been used to demonstrate the existence of gp 145.sup.trkB and gp95.sup.trkB in adult mouse brain (Klein et al., (1990)).
Cross-linking and binding experiments have been used to study the requisite interaction between neurotrophic factors and specific cell surface receptors (Chao, (1990)). Two binding affinities, one high, the other low, have been described for NGF (A. Sutter et al., J. Biol. Chem. 254:5972-5982 (1979)) and BDNF (A. Rodriguez-Tebar and Y.-A. Barde, Nature 8:3337-3342 (1988)) in sensory neurons, although the molecular nature of the high binding affinity has remained obscure. A cDNA encoding a 75 kd (gp75.sup.NGFR) receptor that binds NGF at low affinity has been cloned (D. Johnson et al., Cell 47:545-554 (1986); Radeke et al., (1987)). The recent work of Kaplan et al., (1991 and 1991)) also described in U.S. application Ser. No. 07/668,298 and Klein et al., (1991)) has shed new light on the identity and signal-transducing mechanism of the NGF receptor. Gp 140.sup.proto-trk exhibited low affinity equilibrium binding, while expression of gp75.sup.proto-trk and gp 140.sup.proto-trk in the same cell membrane regenerated a biphasic Scatchard profile similar to that seen in sensory neurons and PC12 cells. Thus, functional high affinity NGF receptors appear to require both gp 75.sup.NGFR and the trk tyrosine kinase receptor (Kaplan et al., (1991); Hempstead et al., (1991)). Applicants have been led by their own prior work to examine whether the evolutionary cousin of trk, trkB, also serves as a receptor for neurotrophic factors (see also D. Soppets et al., Cell 65:985-903 (1991)).
The present invention relates, in part, to a complex comprising NGF ligand and the trk proto-oncogene receptor. It is demonstrated herein that direct binding of NGF to trk receptor leads to tyrosine phosphorylation and tyrosine kinase activity in response to NGF exposure in trk expressing cells. Knowledge of the trk physiological receptor and cognate NGF complex permits nerve growth and regeneration to be studied. Furthermore, the demonstration of NGF-trk receptor complexes offers methods for identifying related tyrosine kinase receptors providing additional neurotrophic-factor pairs. The invention further relates to a similar complex comprising the ligand NT-3 or BDNF complexed with trkB receptor, which complex can be isolated using the methods disclosed herein.